CN112592928B - 冠状病毒的融合基因、融合蛋白、重组载体、通用型dc疫苗及其制备方法 - Google Patents
冠状病毒的融合基因、融合蛋白、重组载体、通用型dc疫苗及其制备方法 Download PDFInfo
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Abstract
本发明提供了冠状病毒的融合基因、融合蛋白、重组载体、通用型DC疫苗及其应用,属于病毒疫苗制备技术领域,融合基因包括编码COVID‑19病毒N蛋白的基因和编码COVID‑19病毒RBD单聚体、二聚体或三聚体蛋白的基因;本发明通过在抗原提呈细胞系中表达融合基因获得一种冠状病毒通用型DC细胞疫苗;所述通用型的DC疫苗可以有效的激活T细胞;根据实施例的记载,本发明制备获得的通用型DC疫苗可以有效激活食蟹猴针对COVID‑19病毒N蛋白和RBD蛋白的特异性的抗体。
Description
技术领域
本发明属于病毒疫苗制备技术领域,尤其涉及三种冠状病毒的融合基因、融合蛋白、重组载体、通用型DC疫苗及其制备方法。
背景技术
冠状病毒在系统分类上属冠状病毒科(Coronaviridae)冠状病毒属(Coronavirus),冠状病毒引起的人类疾病主要是呼吸系统感染。目前现有技术中冠状病毒疫苗的种类有灭活冠状病毒疫苗、冠状病毒突起蛋白疫苗和腺病毒载体疫苗等。
但是目前现有技术没有报道关于靶向冠状病毒的通用DC细胞疫苗。
发明内容
有鉴于此,本发明的目的在于提供一种冠状病毒的融合基因、融合蛋白、重组载体、通用型DC疫苗及其制备方法;所述冠状病毒的融合基因包括编码N蛋白的基因分别和编码RBD单聚体、二聚体和三聚体蛋白的基因进行融合;利用所述融合基因制备获得的通用型DC细胞中可以检测到N蛋白和RBD单聚体、二聚体和三聚体蛋白相应的抗原表达,所述通用型的DC疫苗可以有效的刺激特异性T细胞,激活T细胞;通用型DC疫苗可以有效激活食蟹猴针对COVID-19病毒N蛋白和RBD蛋白的特异性的抗体。
为了实现上述发明目的,本发明提供了以下技术方案:
本发明还提供了一种冠状病毒的融合基因,包括编码COVID-19病毒N蛋白的基因和编码COVID-19病毒RBD蛋白的基因;所述编码COVID-19病毒N蛋白的基因和编码COVID-19病毒RBD蛋白的基因通过连接序列连接;
所述编码COVID-19病毒RBD蛋白的基因包括编码RBD蛋白单聚体的基因、编码RBD蛋白二聚体的基因或编码RBD蛋白三聚体的基因。
优选的,所述融合基因的核苷酸序列如SEQ ID No.1~SEQ ID No.3中任意一项所示。
优选的,所述融合蛋白的氨基酸序列如SEQ ID No.4~6任意一项所示。
本发明提供了一种重组载体,包括所述的融合基因和原始表达载体。
优选的,所述原始表达载体为慢病毒载体。
本发明提供了所述的融合基因、所述的融合蛋白、所述的重组载体在制备预防和/或治疗冠状病毒感染引起的疾病的药物中的应用。
本发明提供了一种冠状病毒通用型DC细胞疫苗,通过在抗原提呈细胞系中表达所述的融合基因获得;
所述的抗原提呈细胞系表达TAX和ST40的融合基因;所述TAX和ST40的融合基因的核苷酸序列如SEQ ID No.7所示;
所述的抗原提呈细胞系为有限代数扩增的抗原递呈细胞。
本发明提供了所述的冠状病毒通用型DC细胞疫苗的制备方法,包括以下步骤:
1)将包括TAX和ST40的融合基因的重组载体包装慢病毒,得到包装病毒;
2)将所述包装病毒感染DC细胞获得感染的DC细胞;
3)将所述感染的DC细胞与滋养细胞进行第一共培养4~6周,除去CD3+细胞后,进行第二共培养1~2周,再次除去CD3+细胞后,进行第三共培养2~6个月,收集有限扩增代数的抗原提呈细胞,得到抗原递呈细胞系;
4)将所述表达冠状病毒的融合基因的重组载体转入步骤3)中所述抗原递呈细胞系获得靶向冠状病毒的通用DC细胞疫苗。
优选的,步骤3)中所述感染的DC细胞与滋养细胞的数量比为(0.8~1.2):(0.8~1.2)。
优选的,步骤4)中所述重组载体转入所述抗原递呈细胞系后还包括抗生素筛选步骤。
本发明的有益效果:本发明提供的冠状病毒的融合基因,包括编码COVID-19病毒N蛋白的基因和编码COVID-19病毒RBD蛋白的基因;所述编码COVID-19病毒RBD蛋白的基因包括编码RBA蛋白单聚体的基因、编码RBD蛋白二聚体的基因或编码RBD蛋白三聚体的基因;所述编码COVID-19病毒N蛋白的基因和编码COVID-19病毒RBD单聚体、二聚体或三聚体蛋白的基因通过连接序列连接。利用所述融合基因制备获得的通用型DC中可以检测到N蛋白和RBD蛋白相应的抗原表达,所述通用型的DC疫苗可以有效的刺激特异性T细胞,激活T细胞;根据实施例的记载,本发明制备获得的通用型DC疫苗可以有效激活食蟹猴针对COVID-19病毒N蛋白和RBD蛋白的特异性的抗体。
附图说明
图1为Western鉴定抗原表达结果;
图2为流式检测通用型DC激活T细胞比例,其中从左至右,从上到下依次为Control、N-RBD-monomer、N-RBD-dimer和N-RBD-trimer;
图3为食蟹猴血清中针对N蛋白的病毒抗原特异性IgG抗体。
图4为食蟹猴血清中针对RBD蛋白的病毒抗原特异性IgG抗体。
具体实施方式
本发明提供了冠状病毒的融合基因,包括编码COVID-19病毒N蛋白的基因和编码COVID-19病毒RBD蛋白的基因;所述编码COVID-19病毒N蛋白的基因和编码COVID-19病毒RBD蛋白的基因通过连接序列连接;所述编码COVID-19病毒RBD蛋白的基因包括编码RBD蛋白单聚体的基因、编码RBD蛋白二聚体的基因或编码RBD蛋白三聚体的基因。
在本发明中,所述融合基因的核苷酸序列如SEQ ID No.1~SEQ ID No.3中任意一项所示。在本发明中,SEQ ID No.1所示的融合基因包括编码COVID-19病毒N蛋白的基因和编码COVID-19病毒RBD蛋白单聚体的基因;SEQ ID No.2所示的融合基因包括编码COVID-19病毒N蛋白的基因和编码COVID-19病毒RBD蛋白二聚体的基因;SEQ ID No.3所示的融合基因包括编码COVID-19病毒N蛋白的基因和编码COVID-19病毒RBD蛋白三聚体的基因;具体序列信息如下:
N-RBD-monomer的核苷酸序列(SEQ ID No.1):
N-RBD-dimer的核苷酸序列(SEQ ID No.2):
N-RBD-trimer的核苷酸序列(SEQ ID No.3):
本发明还提供了所述的融合基因编码的融合蛋白,所述融合蛋白的氨基酸序列如SEQ ID No.4~6所示,具体序列信息如下:
N-RBD-monomer氨基酸序列(SEQ ID No.4):
N-RBD-dimer氨基酸序列(SEQ ID No.5):
N-RBD-trimer氨基酸序列(SEQ ID No.6):
在本发明中,所述编码COVID-19病毒N蛋白的基因的核苷酸序列优选的经过密码子优化,如SEQ ID No.8所示,所述编码COVID-19病毒RBD蛋白单聚体基因的核苷酸序列优选经过密码子优化,如SEQ ID No.9所示,所述编码COVID-19病毒RBD蛋白二聚体基因的核苷酸序列优选经过密码子优化,如SEQ ID No.10所示,具体如下:
agggtgcagccaaccgagtctatcgtgcgctttcctaatatcacaaacctgtgcccatttggcgaggtgttcaacgcaacccgcttcgccagcgtgtacgcctggaataggaagcggatcagcaactgcgtggccgactatagcgtgctgtacaactccgcctctttcagcacctttaagtgctatggcgtgtcccccacaaagctgaatgacctgtgctttaccaacgtctacgccgattctttcgtgatcaggggcgacgaggtgcgccagatcgcccccggccagacaggcaagatcgcagactacaattataagctgccagacgatttcaccggctgcgtgatcgcctggaacagcaacaatctggattccaaagtgggcggcaactacaattatctgtaccggctgtttagaaagagcaatctgaagcccttcgagagggacatctctacagaaatctaccaggccggcagcaccccttgcaatggcgtggagggctttaactgttatttcccactccagtcctacggcttccagcccacaaacggcgtgggctatcagccttaccgcgtggtggtgctgagctttgagctgctgcacgccccagcaacagtgtgcggccccaagaagtccaccaatctggtgaagaacaagtgcgtgaacttcggcagca ccgagttcagcgaggagcagaagaaggccctggacctggccttctacttcgaccgccgcctgacccccgagtggcg ccgctacctgagccagcgcctgggcctgaacgaggagcagatcgagcgctggttccgccgcaaggagcagcagatc ggc;下划线为二聚化结构域。
在本发明中,所述编码COVID-19病毒RBD蛋白三聚体的基因的核苷酸序列优选经过密码子优化,如SEQ ID No.11所示,具体如下:
agggtgcagccaaccgagtctatcgtgcgctttcctaatatcacaaacctgtgcccatttggcgaggtgttcaacgcaacccgcttcgccagcgtgtacgcctggaataggaagcggatcagcaactgcgtggccgactatagcgtgctgtacaactccgcctctttcagcacctttaagtgctatggcgtgtcccccacaaagctgaatgacctgtgctttaccaacgtctacgccgattctttcgtgatcaggggcgacgaggtgcgccagatcgcccccggccagacaggcaagatcgcagactacaattataagctgccagacgatttcaccggctgcgtgatcgcctggaacagcaacaatctggattccaaagtgggcggcaactacaattatctgtaccggctgtttagaaagagcaatctgaagcccttcgagagggacatctctacagaaatctaccaggccggcagcaccccttgcaatggcgtggagggctttaactgttatttcccactccagtcctacggcttccagcccacaaacggcgtgggctatcagccttaccgcgtggtggtgctgagctttgagctgctgcacgccccagcaacagtgtgcggccccaagaagtccaccaatctggtgaagaacaagtgcgtgaacttcggttcag gcggaggttatattcctgaagctccaagagatgggcaagcttacgttcgtaaagatggcgaatgggtattgctttc taccttttta。
本发明还提供了一种重组载体,包括所述的融合基因N-RBD-RBD和原始表达载体。在本发明中,所述原始表达载体为慢病毒载体,更优选为pCDH系列载体或plent-系列载体;所述融合基因优选的重组至所述原始表达载体的XbaI和EcoRI酶切位点之间;所述融合基因优选的由EF1α启动子启动表达。本发明对所述重组载体的制备方法没有特殊限定,采用本领域常规的重组载体的制备方法即可。
本发明提供了所述的融合基因、融合蛋白、重组载体在制备预防和/或治疗冠状病毒感染引起的疾病的药物中的应用。在本发明中,所述冠状病毒为COVID-19病毒;所述药物包括疫苗,优选的包括通用型DC疫苗。
本发明还提供一种冠状病毒通用型DC细胞疫苗,通过在抗原提呈细胞系中表达所述的融合基因获得;所述的抗原提呈细胞系表达TAX和ST40的融合基因;所述TAX和ST40的融合基因的核苷酸序列如SEQ ID No.7所示。
在本发明中,所述TAX基因和ST40基因优选的通过接头序列连接;在本发明中,所述TAX基因的核苷酸序列如SEQ ID NO.12所示,所述ST40基因的核苷酸序列如SEQ IDNO.13所示;所述TAX和ST40的融合基因的核苷酸系列如SEQ ID No.7所示。在本发明中,所述TAX和ST40的融合基因的作用是使树突状细胞永生化,具有有限代次增殖能力。
本发明还提供了所述的冠状病毒通用型DC细胞疫苗的制备方法,包括以下步骤:1)将包括TAX和ST40的融合基因的重组载体包装慢病毒,得到包装病毒;2)将所述包装病毒感染DC细胞获得感染的DC细胞;3)将所述感染的DC细胞与滋养细胞进行第一共培养4~6周,除去CD3+细胞后,进行第二共培养1~2周,再次除去CD3+细胞后,进行第三共培养2~6个月,收集有限扩增代数的抗原提呈细胞,得到抗原递呈细胞系;4)将表达融合基因的重组载体转入步骤3)中所述抗原递呈细胞系获得靶向冠状病毒的通用DC细胞疫苗。
在本发明中,将包括TAX和ST40的融合基因的重组载体包装慢病毒,得到包装病毒。在本发明中,优选的将所述TAX和ST40的融合基因重组至表达载体获得重组载体,所述表达载体优选为制备永生化树突状细胞的质粒(慢病毒载体)pcDH-EF1α-MCS,所述TAX和ST40的融合基因优选的重组至表达载体的EcoRI和BamHI酶切位点之间。本发明对所述包装慢病毒的方法没有特殊限制,采用本领域常规方法即可。
本发明在得到包装病毒后,将所述包装病毒感染DC细胞,得到感染的DC细胞;本发明对所述包装病毒感染DC细胞的方法没有特殊限制,采用本领域常规方法即可。
本发明在得到感染的DC细胞后,将所述感染的DC细胞与滋养细胞进行第一共培养4~6周,除去CD3+细胞后,进行第二共培养1~2周,再次除去CD3+细胞后,进行第三共培养2~6个月,收集有限扩增代数的抗原提呈细胞,得到抗原递呈细胞系。在本发明中,所述感染的DC细胞与滋养细胞优选的在细胞培养器中进行共培养,所述滋养细胞优选的接种于细胞培养器的上层培养室,所述滋养细胞的接种量优选为0.5×106~1.5×106个/孔,更优选为1×106个/孔;所述感染的DC细胞优选的接种于细胞培养器的下层培养室,所述感染的DC细胞的接种量优选为0.5×106~1.5×106个/孔,更优选为1×106个/孔。在本发明中,所述滋养细胞优选的采用以下方法制备得到:将外周血单个核细胞在含有胎牛血清的1640培养基中培养9~14h,优选为12h,得到滋养细胞;所述1640培养基中胎牛血清的质量百分含量为8%~12%,优选为10%。本发明所述抗原递呈细胞能够稳定扩增,能够解决患者自身DC获得困难、不好培养、遗传操作难度较大的问题;并且有限扩增代数,相对于细胞系来说,更安全。
本发明在获得所述抗原递呈细胞系后,将表达基因的重组载体转入所述抗原递呈细胞系获得靶向冠状病毒的通用DC细胞疫苗。本发明对所述转入的具体操作没有特殊限定,采用本领域常规的方法即可。在本发明具体实施过程中,将抗原递呈细胞系与表达基因的重组载体(即表达N-RBD-monomer、N-RBD-dimer、N-RBD-trimer基因的慢病毒)混匀后孵育,所述孵育的温度优选为31~33℃,更优选为32℃,所述孵育的时间优选为4~6h,更优选为5h。本发明在所述重组载体转入所述抗原递呈细胞系后优选的还包括抗生素筛选步骤。在本发明中,所述抗生素优选为嘌呤霉素(2μg/mL)。本发明在所述孵育后,收集细胞,用培养液重悬,与嘌呤霉素混合后,筛选培养4~8天获得靶向冠状病毒的通用DC细胞疫苗。本发明在所述筛选培养期间,维持培养体系中嘌呤霉素的浓度在0.5~2μg/mL之间。
下面结合实施例对本发明提供的技术方案进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
如下实施例中有关试剂配制参见表1。
表1试剂配方
名称 | 配方 |
AP | AIMV中含体积百分比为2.5%FBS, |
1640F | 1640中含体积百分比为10%FBS,250U/mlIL-2 |
AF | AIMV中含体积百分比为10%FBS,250U/mlIL-2 |
实施例1
有限扩增代数的抗原递呈细胞系的建立
1)合成TAX和ST40的融合基因;
2)将步骤1)中合成的融合基因连接到表达载体上获得重组载体;
3)按照慢病毒包装试剂盒(购自于北京合生基因科技有限公司)说明书,将2)所述的载体包装慢病毒,得到包装病毒,将所述包装病毒感染DC的细胞,得到感染的DC的细胞;
4)将滋养细胞接种于细胞培养器的上层培养室,将所述步骤3)得到的感染的DC的细胞接种于细胞培养器的下层培养室,培养4~6周,除去CD3+细胞后继续培养1~2周,除去CD3+细胞后继续培养2个月以上,得到有限扩增代数的抗原提呈细胞系。
DC细胞的分离和活化
1.贴壁法分离外周血来源的树突状细胞(DC)并活化
1)单采血,分离PBMC;
2)将PBMC以培养基1640+10%FBS调整至1×106细胞/mL,至于培养皿中,于37℃、5%CO2培养箱,静置过夜;收集悬浮细胞,标记为T+B,备用;
3)以培养基1640+10%FBS+100IU/mL IL-2对贴在培养皿底部的细胞进行吹打,收集贴壁的单核细胞,即为外周血来源的DC,
4)加入35μg/mL的植物血凝素PHA,刺激并培养细胞24h后备用。
2.融合基因慢病毒表达质粒大提
1)冰上融化转化过融合基因的慢病毒表达质粒的甘油菌stbl3,各取100μl至3ml含有100μg/ml氨苄霉素的LB培养基内,37℃,220rpm活化6h。将菌液分别扩大培养至100ml含有100μg/ml氨苄霉素的LB培养基内,37℃,220rpm培养过夜。次日4000×g离心10min收取各菌液沉淀。
2)准备工作:
a)将RNase A加入Solution I中,4℃保存。
b)在DNA Wash Buffer中加入180ml ethanol(乙醇)。
c)在HBC Buffer中加入76ml isopropanol(异丙醇)。
d)将BufferN3置于冰上,准备42℃水浴。
3)加入3mlGPS Buffer到DNA色谱柱上,室温放置4min,4000×g离心3min,弃废液后备用。
4)取100ml菌沉淀,分别加入10ml Solution I/RNase,上下旋转,吸打混匀。
5)加入10ml Solution II,轻轻反转8~10次,室温3min。
6)加入5ml预冷的BufferN3,轻轻反转8~10次,直至白色絮状沉淀形成,室温2min。
7)利用过滤注射器将上述液体转移至新的50ml离心管内,并测量其体积。
8)加入0.1倍体积的ETR Solution,轻轻反转8~10次。
9)冰上放置10min,期间不断翻转(液体由浑浊变澄清)。
10)42℃水浴5min,液体再次变浑浊。
11)4000×g离心3min,ETR Solution会在底部结成一层。
12)将上清转移至另一新的50ml离心管内,并测量其体积。
13)加入0.5倍体积的酒精,轻轻反转8~10次,室温2min。
14)取20ml上述液体加入到DNA色谱柱内,4000×g离心3min,后弃废液。
15)重复步骤14。
16)向柱内加入10mlHBC Buffer,4000×g离心3min,后弃废液。
17)向柱内加入15ml DNAWash Buffer,4000×g离心3min,后弃废液。
18)向柱内加入10ml DNAWash Buffer,4000×g离心3min,后弃废液。
19)4000×g空离10min后,将色谱柱放入一新的50ml离心管内,向中央膜处滴加800μl dH2O,室温放置5min,4000×g离心5min,将质粒溶液收集到离心管内。
20)将质粒溶液重新加回到柱内,室温放置5min,4000×g离心5min,将质粒溶液重新收集到离心管内,分装冻存。
取个样品2μl,2μl dH2O作为对照,测定浓度。
实施例2构建表达ST40-TAX2融合基因的慢病毒载体并转化DC
1)包装慢病毒:293T细胞铺板密度:1.8×107,20mL OPTI-MEM培养基置于37℃5%CO2培养。实施例1得到的转染质粒用量:包装质粒用量:15μg,P-ST40-TAX2质粒(制备永生化树突状细胞的质粒):15μg,将质粒加入到缓冲液中震荡混匀5s,加入转染试剂:60μL,移液器混匀5下,室温孵育10min,将转染混合物均匀分布滴入细胞中,37℃5%CO2培养。转染后3h,更换新鲜培养基,每个T175的培养瓶加入37mL OPTI-MEM(6%FBS)培养基,96h收获上清后加入病毒浓缩液(购自北京合生基因科技有限公司),沉淀24h后,4000g离心30min,收获病毒,备用。
2)慢病毒感染DC:使用1mL 1640+10%FBS+0.1%polybrene+100μL病毒上清将实施例1中得到的外周血来源的DC,按照3×105个/孔加入到6孔板中,32℃孵育4~6h后放入CO2培养箱培养,4h后补加2mL 1640+10%FBS。培养至第3天时转移至插孔式细胞培养器的下层,标记为Day 0。
3)将实施例1中得到的T+B细胞,以1640+10%FBS+100IU/mL的IL-2重悬,作为滋养细胞加入到插入式细胞培养器(12孔板)的上层培养室,接入细胞数为1×106细胞/孔;
4)置于37℃5%CO2培养箱中,培养,每天观察下层细胞状态,当细胞液变黄时,进行半量换液;
5)每天对上层细胞,进行计数及活率检测,当上层滋养细胞的活率低于50%时,更换新的滋养细胞;
6)根据生长情况,将细胞进行转接,转接的细胞数与起始一致。
a)培养4~6周时,收集所有下层细胞,利用美天旎的CD3+分选磁珠,进行CD3+细胞的去除;
b)将剩余细胞,以1640+10%FBS+100IU/mL IL-2重悬,置于37℃5%CO2培养箱中,培养1~2周;
c)以流式细胞分析仪对细胞表型进行检测,如还有CD3+细胞,则可以重复以上分离步骤;如果没有CD3+细胞,以1640+10%FBS+100IU/mL IL-2培养基中继续培养,2个月后,即可得到通用的DC。
实施例3靶向冠状病毒N蛋白和RBD蛋白的通用型DC疫苗的构建
1)委托生物技术公司合成冠状病毒N蛋白+RBD单聚体、N蛋白+RBD二聚体、N蛋白+RBD三聚体蛋白融合基因;
2)将合成的融合基因连接到慢病毒表达载体上获得表达N-RBD-monomer、N-RBD-dimer和N-RBD-trimer基因的重组载体,包装慢病毒,分装后-80℃冰箱保存;
3)将步骤2)所述重组载体转入实施例2中制备得到的通用型抗原递呈细胞,加入嘌呤霉素进行筛选,具体步骤如下:
将实施例2中得到的通用DC放置于37℃水浴锅中快速解冻;将其转入15ml离心管中,900rpm离心5min;离心后用10ml重悬,计数;取12孔板,按照1×106/孔进行铺板培养,吸取培养基上清与病毒混匀后,加入对应各孔中;将攻毒后的细胞在32℃温度下孵育4~6h。
细胞培养
第一天:显微镜下观察细胞的状态;攻毒后的悬浮细胞每孔补AF液2ml,放入37℃,5%CO2的培养箱中继续培养;
第三天:用移液管将每孔的细胞转移到15ml离心管中,1000rpm离心5min;离心后弃去上清,取5mlAF培养基重悬,将重悬好的细胞转移至T25培养瓶中,再加入嘌呤霉素(2μg/mL),混匀放入37℃,5%CO2的培养箱中培养;
第五天:每瓶补7mlAF培养基,和嘌呤霉素(2μg/mL);
第六天:细胞进行扩瓶培养,用移液管将细胞转移至50ml离心管中,600rpm离心5min;离心后弃上清,取20~30ml 1640F培养基重悬,将重悬好的细胞放入T75培养瓶中,加入嘌呤霉素(2μg/mL),摇晃均匀放入37℃,5%CO2的培养箱中培养;
第八天:细胞进行扩瓶培养。用移液管将细胞转移至50ml离心管中,600rpm离心5min;离心后弃上清,取50ml 1640F培养基重悬,将重悬好的细胞放入T75培养瓶中,加入嘌呤霉素(2μg/mL),摇晃均匀放入37℃,5%CO2的培养箱中培养;
第十天:细胞进行扩瓶培养。用移液管将细胞转移至50ml离心管中,900rpm离心5min.;离心后弃上清,取1640F培养基重悬,将重悬好的细胞放入1个T175(200ml/瓶)培养瓶中,摇晃均匀放入37℃,5%CO2的培养箱中培养;
取此细胞,进行基因组的提取,以基因组为模板,利用PCR的方扩增融合基因序列,然后进行sanger测序,测序正确后,即为针对冠状病毒融合蛋白的通用型DC细胞疫苗。
然后收集N-RBD-monmer-DC、N-RBD-dimer-DC、N-RBD-trimer-DC和DC细胞,将细胞样本以4℃预冷PBS离心两次,2500xg,5min,以每2×106个细胞加入200μl RIPA裂解液(RIPA裂解液使用前加入适当体积HaltTMProtease Inhibitor Cocktail),冰上裂解15min,每隔5min震荡30sec,4℃预冷离心机,细胞裂解液以14000×g离心15min,转移上清至新离心管中,标记后-80℃保存。从-80℃中取出样品溶解,加入适当体积6×Protein LoadingBuffer混匀,100℃沸水浴10min,取出冷却后分装。取样本进行Western Blot检测抗原的表达情况,结果如图1所示:N-RBD-monomer-DC、N-RBD-dimer-DC和N-RBD-trimer-DC中N蛋白表达量基本一致,RBD蛋白量的表达量,呈递增趋势,与预期结果一致。
1.病毒抗原通用型DC细胞激活T细胞作用
以通用型DC细胞疫苗刺激培养CTL后,再加入通用型DC细胞进行二次刺激,并检测CD137的表达情况,结果图2所示,其中Control为无通用型DC细胞刺激培养的细胞,结果表明:N-RBD-monomer-DC、N-RBD-dimer-DC和N-RBD-trimer-DC激活CD8+T细胞的比例分别为:8.05%、13.5%和27.9%,表明这3种组合的二价通用型DC均可有效激活T细胞。
2.食蟹猴安全性评价
食蟹猴重复皮下注射给予N-RBD-monomer-DC、N-RBD-dimer-DC和N-RBD-trimer-DC,2周给药1次,连续给药,共给药2次。即于D1和D15、进行给药。选择开始年龄约为2~5岁,体重约为2.5~5kg的普通级养殖食蟹猴,共使用6只,雌雄各半,分为3组:注射剂量5×107cell/只(最高剂量相当于临床计划给药高剂量的50倍)(下表2)。观察其免疫原性情况和初步安全性情况。
表2检测试验动物分组情况
一般临床观察结果显示,注射通用型DC疫苗后,1~3组食蟹猴精神状态良好,行为外观无明显异常;体重及体温未见明显异常;临床病理检测结果显示,除3外,其他动物接种后,WBC有不同程度的升高,属于正常免疫反应,其余指标未见明显异常。血液生化检测结果显示1~3组动物凝血功能及血生化指标未见明显异常。
IgG抗体检测
分别在首次药前、D7、D14、D21和D28采集血清。检测血清中针对新型冠状病毒N蛋白和RBD蛋白抗原特异性的IgG抗体的变化。
检测结果如图3和4所示,图3为食蟹猴血清中针对N蛋白的病毒抗原特异性IgG抗体,图4为食蟹猴血清中针对RBD蛋白的病毒抗原特异性IgG抗体;1~3组动物中,在1周、2周时与接种前相比,病毒抗原特异性的IgG抗体均有不同程度的增加,第二次免疫后,6只食蟹猴第3周的血清中,针对N蛋白和RBD蛋白的病毒抗原特异性的IgG显著升高。在RBD二聚体和三聚体组中,针对N蛋白和RBD-RBD蛋白的病毒特异性IgG抗体显著升高。表明通用型DC疫苗可以有效激活食蟹猴针对COVID-19病毒N蛋白和RBD蛋白的特异性的抗体。详细滴度见表3和表4。
表3 N特异性IgG抗体滴度检测
表4 RBD特异性IgG抗体滴度检测
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 北京鼎成肽源生物技术有限公司
焦顺昌
<120> 冠状病毒的融合基因、融合蛋白、重组载体、通用型DC疫苗及其制备方法
<160> 13
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1989
<212> DNA
<213> Artificial Sequence
<400> 1
atgtctgaca acggccctca gaaccagcgg aatgccccaa gaatcacctt cggcggcccc 60
tccgattcta caggctccaa ccagaatgga gagaggtccg gagcacgctc taagcagcgg 120
agaccacagg gcctgcccaa caataccgcc agctggttca ccgccctgac acagcacggc 180
aaggaggacc tgaagtttcc caggggccag ggcgtgccta tcaacaccaa tagctcccct 240
gacgatcaga tcggctacta taggagggca acaaggagaa tccggggagg cgacggcaag 300
atgaaggatc tgtcccccag atggtacttc tactatctgg gaaccggacc tgaggcagga 360
ctgccatatg gcgccaataa ggacggaatc atctgggtgg caaccgaggg cgccctgaac 420
acaccaaagg atcacatcgg cacacgcaat cccgccaaca atgcagcaat cgtgctgcag 480
ctgccacagg gaaccacact gcccaagggc ttttacgcag agggcagcag gggaggctcc 540
caggcctcta gccgctcctc tagccggtcc agaaactcct ctcggaacag caccccaggc 600
agctccaggg gcacaagccc tgcaagaatg gcaggaaacg gaggcgacgc cgccctggcc 660
ctgctgctgc tggatagact gaatcagctg gagtctaaga tgagcggcaa gggacagcag 720
cagcagggac agaccgtgac aaagaagtct gccgccgagg ccagcaagaa gccaaggcag 780
aagcgcaccg ccacaaaggc ctacaacgtg acccaggcct tcggcaggcg cggaccagag 840
cagacacagg gcaattttgg cgaccaggag ctgatcaggc agggcaccga ttataagcac 900
tggcctcaga tcgcacagtt cgcaccaagc gcctccgcct tctttggcat gagcaggatc 960
ggaatggagg tgaccccatc cggcacatgg ctgacctaca caggcgccat caagctggac 1020
gataaggacc ctaacttcaa ggatcaggtc atcctgctga acaagcacat cgatgcctat 1080
aagacctttc cccctacaga gcccaagaag gacaagaaga agaaggccga tgagacccag 1140
gccctgcctc agagacagaa gaagcagcag accgtgacac tgctgccagc agcagacctg 1200
gacgattttt ccaagcagct gcagcagtct atgtctagcg ccgatagcac ccaggccgga 1260
tccatggaag gtagaggttc tctcctcact tgtggtgatg ttgaagaaaa ccctggtcca 1320
agggtgcagc caaccgagtc tatcgtgcgc tttcctaata tcacaaacct gtgcccattt 1380
ggcgaggtgt tcaacgcaac ccgcttcgcc agcgtgtacg cctggaatag gaagcggatc 1440
agcaactgcg tggccgacta tagcgtgctg tacaactccg cctctttcag cacctttaag 1500
tgctatggcg tgtcccccac aaagctgaat gacctgtgct ttaccaacgt ctacgccgat 1560
tctttcgtga tcaggggcga cgaggtgcgc cagatcgccc ccggccagac aggcaagatc 1620
gcagactaca attataagct gccagacgat ttcaccggct gcgtgatcgc ctggaacagc 1680
aacaatctgg attccaaagt gggcggcaac tacaattatc tgtaccggct gtttagaaag 1740
agcaatctga agcccttcga gagggacatc tctacagaaa tctaccaggc cggcagcacc 1800
ccttgcaatg gcgtggaggg ctttaactgt tatttcccac tccagtccta cggcttccag 1860
cccacaaacg gcgtgggcta tcagccttac cgcgtggtgg tgctgagctt tgagctgctg 1920
cacgccccag caacagtgtg cggccccaag aagtccacca atctggtgaa gaacaagtgc 1980
gtgaacttc 1989
<210> 2
<211> 2151
<212> DNA
<213> Artificial Sequence
<400> 2
atgtctgaca acggccctca gaaccagcgg aatgccccaa gaatcacctt cggcggcccc 60
tccgattcta caggctccaa ccagaatgga gagaggtccg gagcacgctc taagcagcgg 120
agaccacagg gcctgcccaa caataccgcc agctggttca ccgccctgac acagcacggc 180
aaggaggacc tgaagtttcc caggggccag ggcgtgccta tcaacaccaa tagctcccct 240
gacgatcaga tcggctacta taggagggca acaaggagaa tccggggagg cgacggcaag 300
atgaaggatc tgtcccccag atggtacttc tactatctgg gaaccggacc tgaggcagga 360
ctgccatatg gcgccaataa ggacggaatc atctgggtgg caaccgaggg cgccctgaac 420
acaccaaagg atcacatcgg cacacgcaat cccgccaaca atgcagcaat cgtgctgcag 480
ctgccacagg gaaccacact gcccaagggc ttttacgcag agggcagcag gggaggctcc 540
caggcctcta gccgctcctc tagccggtcc agaaactcct ctcggaacag caccccaggc 600
agctccaggg gcacaagccc tgcaagaatg gcaggaaacg gaggcgacgc cgccctggcc 660
ctgctgctgc tggatagact gaatcagctg gagtctaaga tgagcggcaa gggacagcag 720
cagcagggac agaccgtgac aaagaagtct gccgccgagg ccagcaagaa gccaaggcag 780
aagcgcaccg ccacaaaggc ctacaacgtg acccaggcct tcggcaggcg cggaccagag 840
cagacacagg gcaattttgg cgaccaggag ctgatcaggc agggcaccga ttataagcac 900
tggcctcaga tcgcacagtt cgcaccaagc gcctccgcct tctttggcat gagcaggatc 960
ggaatggagg tgaccccatc cggcacatgg ctgacctaca caggcgccat caagctggac 1020
gataaggacc ctaacttcaa ggatcaggtc atcctgctga acaagcacat cgatgcctat 1080
aagacctttc cccctacaga gcccaagaag gacaagaaga agaaggccga tgagacccag 1140
gccctgcctc agagacagaa gaagcagcag accgtgacac tgctgccagc agcagacctg 1200
gacgattttt ccaagcagct gcagcagtct atgtctagcg ccgatagcac ccaggccgga 1260
tccatggaag gtagaggttc tctcctcact tgtggtgatg ttgaagaaaa ccctggtcca 1320
agggtgcagc caaccgagtc tatcgtgcgc tttcctaata tcacaaacct gtgcccattt 1380
ggcgaggtgt tcaacgcaac ccgcttcgcc agcgtgtacg cctggaatag gaagcggatc 1440
agcaactgcg tggccgacta tagcgtgctg tacaactccg cctctttcag cacctttaag 1500
tgctatggcg tgtcccccac aaagctgaat gacctgtgct ttaccaacgt ctacgccgat 1560
tctttcgtga tcaggggcga cgaggtgcgc cagatcgccc ccggccagac aggcaagatc 1620
gcagactaca attataagct gccagacgat ttcaccggct gcgtgatcgc ctggaacagc 1680
aacaatctgg attccaaagt gggcggcaac tacaattatc tgtaccggct gtttagaaag 1740
agcaatctga agcccttcga gagggacatc tctacagaaa tctaccaggc cggcagcacc 1800
ccttgcaatg gcgtggaggg ctttaactgt tatttcccac tccagtccta cggcttccag 1860
cccacaaacg gcgtgggcta tcagccttac cgcgtggtgg tgctgagctt tgagctgctg 1920
cacgccccag caacagtgtg cggccccaag aagtccacca atctggtgaa gaacaagtgc 1980
gtgaacttcg gcagcaccga gttcagcgag gagcagaaga aggccctgga cctggccttc 2040
tacttcgacc gccgcctgac ccccgagtgg cgccgctacc tgagccagcg cctgggcctg 2100
aacgaggagc agatcgagcg ctggttccgc cgcaaggagc agcagatcgg c 2151
<210> 3
<211> 2082
<212> DNA
<213> Artificial Sequence
<400> 3
atgtctgaca acggccctca gaaccagcgg aatgccccaa gaatcacctt cggcggcccc 60
tccgattcta caggctccaa ccagaatgga gagaggtccg gagcacgctc taagcagcgg 120
agaccacagg gcctgcccaa caataccgcc agctggttca ccgccctgac acagcacggc 180
aaggaggacc tgaagtttcc caggggccag ggcgtgccta tcaacaccaa tagctcccct 240
gacgatcaga tcggctacta taggagggca acaaggagaa tccggggagg cgacggcaag 300
atgaaggatc tgtcccccag atggtacttc tactatctgg gaaccggacc tgaggcagga 360
ctgccatatg gcgccaataa ggacggaatc atctgggtgg caaccgaggg cgccctgaac 420
acaccaaagg atcacatcgg cacacgcaat cccgccaaca atgcagcaat cgtgctgcag 480
ctgccacagg gaaccacact gcccaagggc ttttacgcag agggcagcag gggaggctcc 540
caggcctcta gccgctcctc tagccggtcc agaaactcct ctcggaacag caccccaggc 600
agctccaggg gcacaagccc tgcaagaatg gcaggaaacg gaggcgacgc cgccctggcc 660
ctgctgctgc tggatagact gaatcagctg gagtctaaga tgagcggcaa gggacagcag 720
cagcagggac agaccgtgac aaagaagtct gccgccgagg ccagcaagaa gccaaggcag 780
aagcgcaccg ccacaaaggc ctacaacgtg acccaggcct tcggcaggcg cggaccagag 840
cagacacagg gcaattttgg cgaccaggag ctgatcaggc agggcaccga ttataagcac 900
tggcctcaga tcgcacagtt cgcaccaagc gcctccgcct tctttggcat gagcaggatc 960
ggaatggagg tgaccccatc cggcacatgg ctgacctaca caggcgccat caagctggac 1020
gataaggacc ctaacttcaa ggatcaggtc atcctgctga acaagcacat cgatgcctat 1080
aagacctttc cccctacaga gcccaagaag gacaagaaga agaaggccga tgagacccag 1140
gccctgcctc agagacagaa gaagcagcag accgtgacac tgctgccagc agcagacctg 1200
gacgattttt ccaagcagct gcagcagtct atgtctagcg ccgatagcac ccaggccgga 1260
tccatggaag gtagaggttc tctcctcact tgtggtgatg ttgaagaaaa ccctggtcca 1320
agggtgcagc caaccgagtc tatcgtgcgc tttcctaata tcacaaacct gtgcccattt 1380
ggcgaggtgt tcaacgcaac ccgcttcgcc agcgtgtacg cctggaatag gaagcggatc 1440
agcaactgcg tggccgacta tagcgtgctg tacaactccg cctctttcag cacctttaag 1500
tgctatggcg tgtcccccac aaagctgaat gacctgtgct ttaccaacgt ctacgccgat 1560
tctttcgtga tcaggggcga cgaggtgcgc cagatcgccc ccggccagac aggcaagatc 1620
gcagactaca attataagct gccagacgat ttcaccggct gcgtgatcgc ctggaacagc 1680
aacaatctgg attccaaagt gggcggcaac tacaattatc tgtaccggct gtttagaaag 1740
agcaatctga agcccttcga gagggacatc tctacagaaa tctaccaggc cggcagcacc 1800
ccttgcaatg gcgtggaggg ctttaactgt tatttcccac tccagtccta cggcttccag 1860
cccacaaacg gcgtgggcta tcagccttac cgcgtggtgg tgctgagctt tgagctgctg 1920
cacgccccag caacagtgtg cggccccaag aagtccacca atctggtgaa gaacaagtgc 1980
gtgaacttcg gttcaggcgg aggttatatt cctgaagctc caagagatgg gcaagcttac 2040
gttcgtaaag atggcgaatg ggtattgctt tctacctttt ta 2082
<210> 4
<211> 663
<212> PRT
<213> Artificial Sequence
<400> 4
Met Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg Ile Thr
1 5 10 15
Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg
20 25 30
Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn
35 40 45
Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu
50 55 60
Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro
65 70 75 80
Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly
85 90 95
Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr
100 105 110
Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp
115 120 125
Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp
130 135 140
His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln
145 150 155 160
Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser
165 170 175
Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn
180 185 190
Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr Ser Pro Ala
195 200 205
Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu
210 215 220
Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln
225 230 235 240
Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys
245 250 255
Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln
260 265 270
Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp
275 280 285
Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile
290 295 300
Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile
305 310 315 320
Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Gly Ala
325 330 335
Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile Leu
340 345 350
Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro
355 360 365
Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu Pro Gln
370 375 380
Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala Asp Leu
385 390 395 400
Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala Asp Ser
405 410 415
Thr Gln Ala Gly Ser Met Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly
420 425 430
Asp Val Glu Glu Asn Pro Gly Pro Arg Val Gln Pro Thr Glu Ser Ile
435 440 445
Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe
450 455 460
Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile
465 470 475 480
Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe
485 490 495
Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu
500 505 510
Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu
515 520 525
Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn
530 535 540
Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser
545 550 555 560
Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg
565 570 575
Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr
580 585 590
Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe
595 600 605
Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly
610 615 620
Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu
625 630 635 640
His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val
645 650 655
Lys Asn Lys Cys Val Asn Phe
660
<210> 5
<211> 717
<212> PRT
<213> Artificial Sequence
<400> 5
Met Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg Ile Thr
1 5 10 15
Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg
20 25 30
Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn
35 40 45
Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu
50 55 60
Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro
65 70 75 80
Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly
85 90 95
Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr
100 105 110
Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp
115 120 125
Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp
130 135 140
His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln
145 150 155 160
Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser
165 170 175
Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn
180 185 190
Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr Ser Pro Ala
195 200 205
Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu
210 215 220
Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln
225 230 235 240
Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys
245 250 255
Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln
260 265 270
Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp
275 280 285
Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile
290 295 300
Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile
305 310 315 320
Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Gly Ala
325 330 335
Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile Leu
340 345 350
Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro
355 360 365
Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu Pro Gln
370 375 380
Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala Asp Leu
385 390 395 400
Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala Asp Ser
405 410 415
Thr Gln Ala Gly Ser Met Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly
420 425 430
Asp Val Glu Glu Asn Pro Gly Pro Arg Val Gln Pro Thr Glu Ser Ile
435 440 445
Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe
450 455 460
Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile
465 470 475 480
Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe
485 490 495
Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu
500 505 510
Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu
515 520 525
Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn
530 535 540
Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser
545 550 555 560
Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg
565 570 575
Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr
580 585 590
Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe
595 600 605
Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly
610 615 620
Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu
625 630 635 640
His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val
645 650 655
Lys Asn Lys Cys Val Asn Phe Gly Ser Thr Glu Phe Ser Glu Glu Gln
660 665 670
Lys Lys Ala Leu Asp Leu Ala Phe Tyr Phe Asp Arg Arg Leu Thr Pro
675 680 685
Glu Trp Arg Arg Tyr Leu Ser Gln Arg Leu Gly Leu Asn Glu Glu Gln
690 695 700
Ile Glu Arg Trp Phe Arg Arg Lys Glu Gln Gln Ile Gly
705 710 715
<210> 6
<211> 694
<212> PRT
<213> Artificial Sequence
<400> 6
Met Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg Ile Thr
1 5 10 15
Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg
20 25 30
Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn
35 40 45
Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu
50 55 60
Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro
65 70 75 80
Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly
85 90 95
Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr
100 105 110
Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp
115 120 125
Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp
130 135 140
His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln
145 150 155 160
Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser
165 170 175
Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn
180 185 190
Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr Ser Pro Ala
195 200 205
Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu
210 215 220
Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln
225 230 235 240
Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys
245 250 255
Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln
260 265 270
Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp
275 280 285
Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile
290 295 300
Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile
305 310 315 320
Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Gly Ala
325 330 335
Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile Leu
340 345 350
Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro
355 360 365
Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu Pro Gln
370 375 380
Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala Asp Leu
385 390 395 400
Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala Asp Ser
405 410 415
Thr Gln Ala Gly Ser Met Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly
420 425 430
Asp Val Glu Glu Asn Pro Gly Pro Arg Val Gln Pro Thr Glu Ser Ile
435 440 445
Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe
450 455 460
Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile
465 470 475 480
Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe
485 490 495
Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu
500 505 510
Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu
515 520 525
Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn
530 535 540
Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser
545 550 555 560
Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg
565 570 575
Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr
580 585 590
Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe
595 600 605
Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly
610 615 620
Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu
625 630 635 640
His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val
645 650 655
Lys Asn Lys Cys Val Asn Phe Gly Ser Gly Gly Gly Tyr Ile Pro Glu
660 665 670
Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val
675 680 685
Leu Leu Ser Thr Phe Leu
690
<210> 7
<211> 996
<212> DNA
<213> Artificial Sequence
<400> 7
atgaacatca aagacgaatg gtactggggt aagagtaagc acgcggtgac tgagctcaac 60
gcggagggat ggatctttac tctcccgcca agtgacaact acatcggacg tcaccggttg 120
ccggacgtcc gattcagcca ggagctaccc gacgggacgg tctactggtc ggtgaaccgg 180
aagaacttct tccgccggga cgacagcctc ccctcgggat gggtgcagcg catctacccg 240
cgtgtagcta ccagcttcag gaccgcggaa tgagccacga acttctctct gttaaagcaa 300
gcaggagatg ttgaagaaaa ccccgggcct gcccatttcc caggatttgg acagagcctc 360
ctatatggat accccgtcta cgtgtttggc gattgtgtac aggccgattg gtgtcccgtc 420
tcaggtggtc tatgttccac ccgcctacat cgacatgccc tcctggccac ctgtccagag 480
caccaactca cctgggaccc catcgatgga cgcgttgtca gctctcctct ccaatacctt 540
atccctcgcc tcccctcctt ccccacccag agaacctcaa ggaccctcaa ggtccttacc 600
cctcccacca ctcctgtctc ccccaaggtt ccacctgcct tctttcaatc aatgcgaaag 660
cacaccccct accgaaatgg atgcctggaa ccaaccctcg gggatcagct cccctccctc 720
gccttccccg aacctggcct ccgtccccaa aacatctaca ccacctgggg aaaaaccgta 780
gtatgcctat acctatacca gctttcccca cccatgacat ggccacttat accccatgtc 840
atattctgcc accccagaca attaggagcc ttcctcacca aggtgcctct aaaacgatta 900
gaagaacttc tatacaaaat gttcctacac acagggacag tcatagtcct cccggaggac 960
gacctaccca ccacaatgtt ccaacccgtg agggct 996
<210> 8
<211> 1257
<212> DNA
<213> Artificial Sequence
<400> 8
atgtctgaca acggccctca gaaccagcgg aatgccccaa gaatcacctt cggcggcccc 60
tccgattcta caggctccaa ccagaatgga gagaggtccg gagcacgctc taagcagcgg 120
agaccacagg gcctgcccaa caataccgcc agctggttca ccgccctgac acagcacggc 180
aaggaggacc tgaagtttcc caggggccag ggcgtgccta tcaacaccaa tagctcccct 240
gacgatcaga tcggctacta taggagggca acaaggagaa tccggggagg cgacggcaag 300
atgaaggatc tgtcccccag atggtacttc tactatctgg gaaccggacc tgaggcagga 360
ctgccatatg gcgccaataa ggacggaatc atctgggtgg caaccgaggg cgccctgaac 420
acaccaaagg atcacatcgg cacacgcaat cccgccaaca atgcagcaat cgtgctgcag 480
ctgccacagg gaaccacact gcccaagggc ttttacgcag agggcagcag gggaggctcc 540
caggcctcta gccgctcctc tagccggtcc agaaactcct ctcggaacag caccccaggc 600
agctccaggg gcacaagccc tgcaagaatg gcaggaaacg gaggcgacgc cgccctggcc 660
ctgctgctgc tggatagact gaatcagctg gagtctaaga tgagcggcaa gggacagcag 720
cagcagggac agaccgtgac aaagaagtct gccgccgagg ccagcaagaa gccaaggcag 780
aagcgcaccg ccacaaaggc ctacaacgtg acccaggcct tcggcaggcg cggaccagag 840
cagacacagg gcaattttgg cgaccaggag ctgatcaggc agggcaccga ttataagcac 900
tggcctcaga tcgcacagtt cgcaccaagc gcctccgcct tctttggcat gagcaggatc 960
ggaatggagg tgaccccatc cggcacatgg ctgacctaca caggcgccat caagctggac 1020
gataaggacc ctaacttcaa ggatcaggtc atcctgctga acaagcacat cgatgcctat 1080
aagacctttc cccctacaga gcccaagaag gacaagaaga agaaggccga tgagacccag 1140
gccctgcctc agagacagaa gaagcagcag accgtgacac tgctgccagc agcagacctg 1200
gacgattttt ccaagcagct gcagcagtct atgtctagcg ccgatagcac ccaggcc 1257
<210> 9
<211> 669
<212> DNA
<213> Artificial Sequence
<400> 9
agggtgcagc caaccgagtc tatcgtgcgc tttcctaata tcacaaacct gtgcccattt 60
ggcgaggtgt tcaacgcaac ccgcttcgcc agcgtgtacg cctggaatag gaagcggatc 120
agcaactgcg tggccgacta tagcgtgctg tacaactccg cctctttcag cacctttaag 180
tgctatggcg tgtcccccac aaagctgaat gacctgtgct ttaccaacgt ctacgccgat 240
tctttcgtga tcaggggcga cgaggtgcgc cagatcgccc ccggccagac aggcaagatc 300
gcagactaca attataagct gccagacgat ttcaccggct gcgtgatcgc ctggaacagc 360
aacaatctgg attccaaagt gggcggcaac tacaattatc tgtaccggct gtttagaaag 420
agcaatctga agcccttcga gagggacatc tctacagaaa tctaccaggc cggcagcacc 480
ccttgcaatg gcgtggaggg ctttaactgt tatttcccac tccagtccta cggcttccag 540
cccacaaacg gcgtgggcta tcagccttac cgcgtggtgg tgctgagctt tgagctgctg 600
cacgccccag caacagtgtg cggccccaag aagtccacca atctggtgaa gaacaagtgc 660
gtgaacttc 669
<210> 10
<211> 831
<212> DNA
<213> Artificial Sequence
<400> 10
agggtgcagc caaccgagtc tatcgtgcgc tttcctaata tcacaaacct gtgcccattt 60
ggcgaggtgt tcaacgcaac ccgcttcgcc agcgtgtacg cctggaatag gaagcggatc 120
agcaactgcg tggccgacta tagcgtgctg tacaactccg cctctttcag cacctttaag 180
tgctatggcg tgtcccccac aaagctgaat gacctgtgct ttaccaacgt ctacgccgat 240
tctttcgtga tcaggggcga cgaggtgcgc cagatcgccc ccggccagac aggcaagatc 300
gcagactaca attataagct gccagacgat ttcaccggct gcgtgatcgc ctggaacagc 360
aacaatctgg attccaaagt gggcggcaac tacaattatc tgtaccggct gtttagaaag 420
agcaatctga agcccttcga gagggacatc tctacagaaa tctaccaggc cggcagcacc 480
ccttgcaatg gcgtggaggg ctttaactgt tatttcccac tccagtccta cggcttccag 540
cccacaaacg gcgtgggcta tcagccttac cgcgtggtgg tgctgagctt tgagctgctg 600
cacgccccag caacagtgtg cggccccaag aagtccacca atctggtgaa gaacaagtgc 660
gtgaacttcg gcagcaccga gttcagcgag gagcagaaga aggccctgga cctggccttc 720
tacttcgacc gccgcctgac ccccgagtgg cgccgctacc tgagccagcg cctgggcctg 780
aacgaggagc agatcgagcg ctggttccgc cgcaaggagc agcagatcgg c 831
<210> 11
<211> 762
<212> DNA
<213> Artificial Sequence
<400> 11
agggtgcagc caaccgagtc tatcgtgcgc tttcctaata tcacaaacct gtgcccattt 60
ggcgaggtgt tcaacgcaac ccgcttcgcc agcgtgtacg cctggaatag gaagcggatc 120
agcaactgcg tggccgacta tagcgtgctg tacaactccg cctctttcag cacctttaag 180
tgctatggcg tgtcccccac aaagctgaat gacctgtgct ttaccaacgt ctacgccgat 240
tctttcgtga tcaggggcga cgaggtgcgc cagatcgccc ccggccagac aggcaagatc 300
gcagactaca attataagct gccagacgat ttcaccggct gcgtgatcgc ctggaacagc 360
aacaatctgg attccaaagt gggcggcaac tacaattatc tgtaccggct gtttagaaag 420
agcaatctga agcccttcga gagggacatc tctacagaaa tctaccaggc cggcagcacc 480
ccttgcaatg gcgtggaggg ctttaactgt tatttcccac tccagtccta cggcttccag 540
cccacaaacg gcgtgggcta tcagccttac cgcgtggtgg tgctgagctt tgagctgctg 600
cacgccccag caacagtgtg cggccccaag aagtccacca atctggtgaa gaacaagtgc 660
gtgaacttcg gttcaggcgg aggttatatt cctgaagctc caagagatgg gcaagcttac 720
gttcgtaaag atggcgaatg ggtattgctt tctacctttt ta 762
<210> 12
<211> 273
<212> DNA
<213> Artificial Sequence
<400> 12
atgaacatca aagacgaatg gtactggggt aagagtaagc acgcggtgac tgagctcaac 60
gcggagggat ggatctttac tctcccgcca agtgacaact acatcggacg tcaccggttg 120
ccggacgtcc gattcagcca ggagctaccc gacgggacgg tctactggtc ggtgaaccgg 180
aagaacttct tccgccggga cgacagcctc ccctcgggat gggtgcagcg catctacccg 240
cgtgtagcta ccagcttcag gaccgcggaa tga 273
<210> 13
<211> 666
<212> DNA
<213> Artificial Sequence
<400> 13
gcccatttcc caggatttgg acagagcctc ctatatggat accccgtcta cgtgtttggc 60
gattgtgtac aggccgattg gtgtcccgtc tcaggtggtc tatgttccac ccgcctacat 120
cgacatgccc tcctggccac ctgtccagag caccaactca cctgggaccc catcgatgga 180
cgcgttgtca gctctcctct ccaatacctt atccctcgcc tcccctcctt ccccacccag 240
agaacctcaa ggaccctcaa ggtccttacc cctcccacca ctcctgtctc ccccaaggtt 300
ccacctgcct tctttcaatc aatgcgaaag cacaccccct accgaaatgg atgcctggaa 360
ccaaccctcg gggatcagct cccctccctc gccttccccg aacctggcct ccgtccccaa 420
aacatctaca ccacctgggg aaaaaccgta gtatgcctat acctatacca gctttcccca 480
cccatgacat ggccacttat accccatgtc atattctgcc accccagaca attaggagcc 540
ttcctcacca aggtgcctct aaaacgatta gaagaacttc tatacaaaat gttcctacac 600
acagggacag tcatagtcct cccggaggac gacctaccca ccacaatgtt ccaacccgtg 660
agggct 666
Claims (9)
1.一种冠状病毒的融合基因,其特征在于,包括编码COVID-19病毒N蛋白的基因和编码COVID-19病毒RBD蛋白的基因;所述编码COVID-19病毒N蛋白的基因和编码COVID-19病毒RBD蛋白的基因通过连接序列连接;
所述编码COVID-19病毒RBD蛋白的基因包括编码RBD蛋白单聚体的基因、编码RBD蛋白二聚体的基因或编码RBD蛋白三聚体的基因;
所述融合基因的核苷酸序列如SEQ ID No.1~SEQ ID No.3中任意一项所示。
2.权利要求1所述的融合基因编码的融合蛋白,其特征在于,所述融合蛋白的氨基酸序列如SEQ ID No.4~6任意一项所示。
3.一种重组载体,其特征在于,包括权利要求1所述的融合基因和原始表达载体。
4.根据权利要求3所述的重组载体,其特征在于,所述原始表达载体为慢病毒载体。
5.权利要求1所述的融合基因、权利要求2所述的融合蛋白、权利要求3或4所述的重组载体在制备预防和/或治疗冠状病毒感染引起的疾病的药物中的应用。
6.一种冠状病毒通用型DC细胞疫苗,其特征在于,通过在抗原提呈细胞系中表达权利要求1中所述的融合基因获得;
所述的抗原提呈细胞系表达TAX和ST40的融合基因;所述TAX和ST40的融合基因的核苷酸序列如SEQ ID No.7所示;
所述的抗原提呈细胞系为有限代数扩增的抗原递呈细胞。
7.权利要求6所述的冠状病毒通用型DC细胞疫苗的制备方法,包括以下步骤:
1)将包括TAX和ST40的融合基因的重组载体包装慢病毒,得到包装病毒;
2)将所述包装病毒感染DC细胞获得感染的DC细胞;
3)将所述感染的DC细胞与滋养细胞进行第一共培养4~6周,除去CD3+细胞后,进行第二共培养1~2周,再次除去CD3+细胞后,进行第三共培养2~6个月,收集有限扩增代数的抗原提呈细胞,得到抗原递呈细胞系;
4)将权利要求3或4中表达冠状病毒的融合基因的重组载体转入步骤3)中所述抗原递呈细胞系获得靶向冠状病毒的通用DC细胞疫苗。
8.根据权利要求7所述的制备方法,其特征在于,步骤3)中所述感染的DC细胞与滋养细胞的数量比为(0.8~1.2):(0.8~1.2)。
9.根据权利要求7所述的制备方法,其特征在于,步骤4)中所述重组载体转入所述抗原递呈细胞系后还包括抗生素筛选步骤。
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